Most of vascular disorders in a brain or a heart are thrombosis, which is mainly caused by abnormality of blood flow, abnormality of coagulation components, and abnormality of a vascular endothelium. Actually, integral action of those abnormalities induces thrombosis. It is thrombin as a serine protease that is involved in all abnormalities and plays a central role in thrombus formation. The thrombin produces a fibrin clump at the final stage of coagulation cascade, and simultaneously accelerates the coagulation cascade by activating XI, V, and VIII factors. In addition, thrombin causes platelet aggregation and activation of endothelial cells through PAR1 which is a thrombin receptor on the platelet and the vascular endothelium. It is reported that the activation of endothelial cells causes the hypercoagulation of a vascular wall and then thrombus formation proceeds, as a result of negative chain Non-patent Document 1).
Non-patent Document 2 describes that an exocite I region plays an important role in substrate recognition which is involved in the main blood coagulation pathway of thrombin. Non-patent Document 3 describes that: a serine protease such as thrombin has serine, histidine, and aspartic acid at the active center; the protease activity is expressed by a charge relay system of those three amino acids; and glycine 193 (the number 193 refers to a position of the amino acid in chymotrypsinogen, and glycine 193 corresponds to glycine at position 203 in thrombin B chain) is involved in the progress from a Michaelis complex to a tetrahedral complex.
Thrombin mutants having amino acid substitutions have been studied. There have been some investigations, as described below, on recombinants in which an amino acid in the active center is replaced as a result of gene recombination of thrombin. For example, Non-patent Document 4 describes the influence of a thrombin mutant, in which serine in the active center is replaced with alanine, on leukocytes. Non-patent Document 5 describes a thrombin mutant in which glycine at position 203 in the B chain is replaced with alanine, a thrombin mutant in which serine in the active center is replaced with alanine or threonine, a thrombin mutant in which histidine in the active center is replaced with asparagine, and a thrombin mutant in which aspartic acid in the active center is replaced with asparagine. However, the thrombin mutants described in Non-patent Documents 4 and 5 do not have sufficient efficiency as an antithrombotic agent or anti-inflammatory agent because of the following reasons: they still have residual enzymatic activity (thrombin substrate-cleaving activity) at a level which cannot be detected by the measurement method described in each of the documents; a thrombin substrate-binding ability thereof is remarkably impaired; or the thrombin mutants have high binding ability to Fbgn which is present in a large amount in blood.
Patent Document 1 and Non-patent Documents 6, 7, and 8 describe thrombin mutants each having an enzymatic activity (thrombin substrate-cleaving activity) and an anti-blood coagulation effect obtained by replacing an amino acid thereof Those thrombin mutants are each a thrombin mutant in which a binding ability to thrombomodulin (hereinafter, may be referred to as “TM”) is maintained or enhanced, a fibrinogen-cleaving ability is remarkably decreased, and an antithrombotic effect is exhibited by binding specifically to TM and activating protein C.
Patent Document 2 discloses a prothrombin derivative which has an amino acid substitution in the active center and is intended to be used for neutralizing an anticoagulation activity of a hirudin C-terminal peptide when problems such as bleeding occurs by administration of the hirudin C-terminal peptide as an antithrombotic agent to a patient.
Patent Documents 3 and 4 describe that a thrombin mutant in which serine in the active center was replaced with alanine and a thrombin mutant in which serine in the active center was replaced with alanine and aspartic acid in the active center was replaced with asparagine inhibited the stimulation of a thrombin receptor by thrombin in a washed platelet suspension.
Patent Document 5 describes various kinds of thrombin mutants which: have lost a substrate-cleaving activity; have decreased affinity to fibrinogen, heparin, and thrombomodulin; and have high antithrombotic ability, as a result of amino acid substitutions in the active center and other sites.
However, the thrombin mutant reported in Patent Document 5 has a little affinity to a heparin-like substance (heparan sulfate), thrombomodulin, and integrin, which are present in a vascular wall, and hence the mutant binds to the vascular wall or the like, a circulating volume thereof in the blood was insufficient, and a half life thereof was short as a result of endocytosis by a vascular endothelium. Thus, there has remained to be improved.
Then, there has been demand for thrombin mutants which have lower affinities to heparin, thrombomodulin, and integlin, and have improved antithrombotic ability.    Patent Document 1: WO 95/13385    Patent Document 2: WO 96/41868    Patent Document 3: WO 92/14750    Patent Document 4: U.S. Pat. No. 5,256,766    Patent Document 5: WO 2005/089070    Non-patent Document 1: J. Biol. Chem. 261 (1986) 15928-15933    Non-patent Document 2: Japanese Journal of Thrombosis and Hemostasis, Vol. 10, Nos. 2 and 3 (1999)    Non-patent Document 3: Voet, Biochemistry, Volume 1, 1996, p. 331-340, TOKYO KAGAKU DOZIN, CO., LTD.    Non-patent Document 4: Experimental cell research, 219, 650-656 (1995)    Non-patent Document 5: Biochimica et BiophysciaActa, 1451 (1999) 173-186    Non-patent Document 6: J. Biol. Chem, Vol. 275, 39827-39830    Non-patent Document 7: J. Biol. Chem, Vol. 279, 26387-26394    Non-patent Document 8: J. Biol. Chem, Vol. 277, 27581-27584